Identify the missing restriction sites (A, B and C) in the given E. c…

The correct answer is Option (1) → A - BamH I; B - Cla I; C - Pst I

pBR322 is an E. coli cloning vector. It carries the ori, the gene rop and two antibiotic resistance genes, amp^R and tet^R, with restriction sites at fixed places. Reading the map clockwise from the top: EcoR I, then B between EcoR I and Hind III, then A inside the tet^R gene before Sal I, and C inside the amp^R gene next to Pvu I.

Option (1) A - BamH I; B - Cla I; C - Pst I — Correct. Cla I sits between EcoR I and Hind III; BamH I lies in the tet^R gene; Pst I lies in the amp^R gene beside Pvu I. A foreign gene put into the BamH I site inactivates tet^R, so the recombinants lose tetracycline resistance.

Option (2) A - Pst I; B - Cla I; C - Hind II — Incorrect. Pst I is in the amp^R gene, not in tet^R. Hind II is not one of the sites marked on pBR322; it is the first restriction endonuclease to be isolated.

Option (3) A - BamH I; B - Hind II; C - Cla I — Incorrect. A is right, but the site next to Hind III is Cla I, not Hind II, and Cla I is not in the amp^R gene.

Option (4) A - Cla I; B - Pst I; C - BamH I — Incorrect. All three are misplaced: BamH I belongs in tet^R, not in amp^R, and Pst I belongs in amp^R, not beside Hind III.

NCERT Extract:
Cloning Vectors – “… (iii) Cloning sites: In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes. Presence of more than one recognition sites within the vector will generate several fragments, which will complicate the gene cloning (Figure 9.4). The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes. For example, you can ligate a foreign DNA at the BamH I site of tetracycline resistance gene in the vector pBR322.”